Poly ADP-ribose synthetase 1 (or PARP-1) is a dimeric nuclear protein, with two 113 kDa polypeptide subunits, each consisting of three functional domains: the DNA binding amino terminal domain with two zinc fingers for recognition of single and double strand DNA breaks, such as those induced by reactive oxygen species (ROS), gamma-irradiation and DNA alkylating agents, the central automodification domain (auto ADP-ribosylation), and the carboxyl terminus catalytic domain, using NAD+ as substrate, for the synthesis of the ADP-ribose polymers, which vary in length between 50-200 subunits. Other family members, all of which share high homology to PARP-1 in the amino and carboxyl termini are PARP-2, 3, vault-PARP and tankyrase. In addition to autocatalytic ADP-ribosylation, PARP-1 has been shown to use histones, topoisomerase I and II, DNA polymerases, and DNA ligase 2 as protein acceptors. This poly ADP-ribosylation appears to inhibit the activity of some of the enzymes, but for histones the ADP-ribosylation has been proposed to stimulate chromosome relaxation allowing for DNA repair. In fact, a requirement for PARP-1 in the recovery from DNA damage induced by N-methyl-N-nitrosourea (MNU) and gamma-radiation was demonstrated using PARP deficient mice and embryo fibroblasts.
Importantly, reactive oxygen species (ROS) have been shown to mediate over-activation of PARP-1, which has been demonstrated to cause critical ATP depletion leading to cell necrosis, as the toxic effects can be substantially ameliorated by either: PARP-1 inhibitors in the cell lineages; macrophages, aortic smooth muscle, neuronal and endothelial, or absence of active PARP-1 in PARP-1−/− deficient fibroblasts.
In addition to the role of PARP-1 in a housekeeping, general genome maintenance function, there is more recent evidence for a role of PARP-1 in specific gene expression, particularly through interaction with Nf-κB. Importantly in the context of ischemia-reperfusion (IR) injury, target genes of Nf-κB in endothelial cells include inducible nitric-oxide synthase (iNOS) and the cell adhesion molecules P-selectin and intracellular adhesion molecule-1 (ICAM-1). Nitric oxide (NO), which is known to have potent vasodilating activity may act as a protective factor during IR injury, however under in the presence of superoxide, endogenous NO has been shown to be detrimental to the health of the IR injured tissues, possibly due to the synthesis of peroxynitrite. In turn, the cell surface expression of P-selectin and ICAM-1 has been shown to mediate the tissue infiltration of neutrophils, which has been demonstrated to contribute to IR-mediated organ damage. PARP-1 deficient mice have been shown to be resistant to the ischemia reperfusion injury of the heart, which is associated with reduced level of ICAM-1 and P-selectin expression in the vascular endothelium and injured myocytes of the myocardium and corresponding neutrophil recruitment induced by ischemia-reperfusion.
Several studies of the use of pharmacological inhibitors of PARP-1 in vivo have demonstrated efficacy in reducing IR induced tissue damage, and improved function of the heart, skeletal muscle, liver and arthritic joints. In a rabbit model, PARP-1 inhibitors significantly decreased infarct size in the heart due to 45-minute occlusion and two-hour reperfusion, as well as skeletal muscle necrosis due to a two-hour occlusion and four-hour reperfusion. In a study of liver microcirculation and function after hemorrhagic shock and resuscitation in rats, the PARP-1 inhibitor 5-aminoisoquinoline (5-AIQ), demonstrating decreased leukocyte-endothelial interaction, decreased liver injury and improved liver function. Also, in a mouse model of arthritis, the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP) reduced the severity of the disease as assessed by the histological parameters; inflammatory cell infiltration, hyperplasia of the synovium and tissue necrosis.
Several studies in animal models of ischemia-reperfusion have been performed specifically to elucidate the possible role of over-activated PARP-1 in the tissue injury. In a mouse model of thoracoabdominal aneurysm mediated renal injury, the potent PARP-1 inhibitor PJ34 was used to determine the possible role of PARP-1 in this setting. Mice were exposed to eleven minutes of aortic ischemia followed by 48 hours of reperfusion, and were treated with PJ34 1 hour before and immediately after the ischemic period. PJ34 was shown to preserve renal mitochondrial activity and decrease steady state levels of a marker for neutrophil infiltration, but had no apparent affect on fibrinolysis stimulated by the ischemia-reperfusion. Studies in a rat model of direct renal ischemia induced by occluding the renal arteries, demonstrated that the PARP-1 inhibitors 3-aminobenzamide (3-AB) and 1,5-dihroxyisoquinoline improved kidney function as measured by blood and urine markers, plasma urea, plasma creatinine and glomerular filtration rate following 45 minutes of ischemia and up to six hours of reperfusion, with the experimental drugs given one minute before reperfusion. Finally, in the study of PARP-1 expression in human transplant recipients between days 5 and 11 of post-transplantation, all with acute tubular necrosis; PARP-1 expression correlated with the duration of cold renal ischemia, with its expression being highest after ten hours and correlating significantly with delayed renal function.
Familial breast and ovarian cancers, which account for 5-10% of these cancers, are commonly caused by the inherited defect in one of the BRCA1 or BRCA2 alleles. During life the normal, functional BRCA1 or BRCA2 alleles can be lost in some cells, thus initiating the development of a tumor. The tumors that developed were BRCA1 or BRCA2 deficient while remaining somatic cells had functional BRCA proteins. As described above, such tumor cells would be expected to be extremely sensitive to PARP-1 inhibition and this has been confirmed recently. Two independent groups demonstrated specific killing of BRCA deficient cells and inhibition of tumor xenograft growth by pharmacological inhibition of PARP-1 alone with no requirement to combine with chemotherapy. In addition cells deficient in other gene products responsible for homologous recombination such as RAD51, RAD54, DSS1, RPA1, NBSI ATM, ATR CHK1, FANCD2, FANCA or FANCC, are also sensitive to PARP-1 inhibition.
Alternatively, for the majority of neoplasias, which are not deficient in HR function; the combination of DNA damaging agents (chemotherapy or radiation treatment) with PARP-1 inhibition would be expected to mimic the PARP-1 deficient animal studies and increase the tumor's sensitivity to the DNA damaging agent, and this has been the case. Synergistic tumor cell killing has been demonstrated using a PARP-1 inhibitor in combination with camptothecin, a topoisomerase I inhibitor; with the PARP-1 inhibitor increasing cytotoxicity and DNA strand breaks in parallel, 2.5 fold. Consistent with this, camptothecin alone induced DNA strand breaks that lead to a four-fold activation of PARP-1. Interestingly, etoposide, a topoisomerase II inhibitor, induced DNA strand breaks but failed to induce PARP-1 or synergize with the PARP-1 inhibitor in cytotoxicity. Similarly, PARP-1 deficient V79 cells were shown to be hypersensitive to topoisomerase I inhibitors but resistant to etoposide. Also, PARP-1 inhibitors could not potentiate the cytotoxicity of cisplatin in ovarian tumor cells, again suggesting specificity to the type of strand breaks. Several studies have shown the potentiation of temozolomide (TMZ) by PARP-1 inhibitors. Studies have demonstrated the potentiation (1.2-5 fold) of TMZ growth inhibition and cytotoxicity by PARP-1 pharmacological inhibition across many tumor cell lines representing lung, ovarian, colon and breast cancers (gliomas not tested in their system). PARP-1 inhibition was shown to increase the antitumor activity of TMZ against intracranial melanoma, lymphoma, and glioma in vivo using murine orthotopic, tumor models; demonstrating improved survival of tumor bearing mice and an increase in anti-metastatic effect of TMZ. Also, other studies have shown that PARP-1 inhibition increased the antiproliferative effect of TMZ in colorectal cancer cell line LoVo by 5.5 fold, and decreased recovery from gamma-radiation damage in this cell line by 75%. In vivo, a non-toxic dose of this inhibitor increased the delay of LoVo xenograft growth induced by irinotecan, TMZ and x-irradiation by two- to threefold. Interestingly, increased antitumor activity was demonstrated from PARP-1 inhibition with TMZ in another colorectal line, SW620 xenografts while no such activity was demonstrated in vitro. Further analysis demonstrated PARP-1 inhibition statistically, significantly increased blood flow to the tumor and thus possibly increased TMZ delivery to the SW620 xenografts. This possible utility of PARP-1 inhibitors in cancer therapy has been described earlier for the less potent PARP-1 inhibitor nicotinamide, which had been shown to inhibit contraction of vascular smooth muscle cells in tumors. Activation of the transcription factor, NF-κB which has been shown in many tumor cell lines, including glioma, in vitro and in vivo to promote cell survival, proliferation, angiogenesis and metastasis, by transcriptional activation of antiapoptotic genes (e.g., cIAP, survivin, Bcl-2 and Bcl-X1) cell cycle regulatory genes (cyclin D1 and c-myc) COX-2, matrix meltalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). As described above, the role of PARP-1 in activation of NF-κB and the benefit of inhibitors of PARP-1 activity also have relevance in cancer therapy.
In certain embodiments, the present invention is directed toward the identification of small organic molecules that exhibit PARP inhibitory activity and are thus useful in the treatment or prevention of conditions or diseases in which inhibition of PARP is desirable.
All citations in the present application are incorporated herein by reference in their entireties. The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.